1. Field of the Invention
The present invention relates to detection of at least one Mycoplasma species, including Mycoplasma fermentans, Mycoplasma hominis and Mycoplasma penetrans, in blood samples of patients with chronic fatigue syndrome, fibromyalgia and rheumatoid arthritis.
2. Description of the Related Art
Chronic Fatigue Syndrome (CFS) is an illness with increasingly reported frequency in the United States and other industrialized countries (Straus, Rev. Infect. Dis. 13(Suppl. 1):S2-S7, 1991). CFS is characterized by prolonged and debilitating fatigue with multiple non-specific symptoms such as headaches, recurring sore throats, muscle and joint pains and cognitive complaints. Profound fatigue, the hallmark of the disorder, can appear suddenly or gradually and persists throughout the course of the illness. Unlike the short-term disability of an acute viral infection, for example, CFS symptoms by definition linger for at least six months and often for years (Fukuda et al., Ann. Intern. Med 121:953-959, 1994). Physicians can evaluate patients with persistent fatigue of undetermined cause using guidelines developed by the international CFS study group (Fukuda et al., Fed. Pract. 12:12-17, 1995).
Despite multidisciplinary investigations of CFS, its etiology remains unknown and no specific diagnostic tests or therapies for CFS exist. In about one third of cases, the sudden onset follows a respiratory, gastrointestinal, or other acute infection with flu-like symptoms, including mononucleosis (Mawle et al., Infect. Agents Dis. 2:333-341, 1994). No published data implicate a specific virus or other microbes as the cause of CFS. However, it appears that infectious agents, among other stressors, can precipitate the syndrome (National Institutes of Health Publication No. 96-484, 1996). A variety of common viruses can be reactivated in some CFS patients, including HTLV-II, Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex viruses (HSV) 1 and 2, and human herpes viruses 6, 7 and 8. It is believed that virus reactivation could be occurring secondarily to some immunologic disturbance (National Institutes of Health Publication No. 96-484, 1996; Nicolson et al., Int. J. Occup. Med. Immunol. Toxicol. 5:69-78, 1996).
It has been well documented that individuals who suffer from fibromyalgia (FMS) exhibit many of the same symptoms found in CFS (Buchwald et al., Arch. Intern. Med. 154:2049-2053, 1994; Ziem et al., Arch. Intern. Med. 154:1913, 1995). These two illnesses are so similar that for years many medical practitioners have considered them to be the same condition. They are still regarded as closely associated with the exception of a few distinction criteria. Patients suffering from rheumatoid arthritis (RA) also exhibit certain symptoms characteristic of CFS and FMS. Although RA exhibits a narrower spectrum of clinical symptoms than the other disorders, it does exhibit a significant overlap of symptoms found in each condition.
Mycoplasmas are bacteria belong to the class Mollicutes. They are the smallest free-living, self-replicating bacteria known. They have no cell wall and a very limited genome of between 600 and 1,500 kilobases which makes them highly dependent on their host for survival. The mycoplasma species M. fermentans, M. hominis and M. penetrans have been isolated from individuals suffering from primary atypical pneumonia, urogenital infections, rheumatoid arthritis (RA) and AIDS-related infections (Hayes et al., Infect. Immun. 64:3419-3424, 1996; Schaeverbeke et al., Br. J. Rheumatol. 36:310-314, 1997; Montagnier et al., Clin. Infect. Dis. 17(Suppl. 1):S309-315, 1993).
Rapid reliable detection techniques are of great importance in a clinical diagnostic setting. Current methods of mycoplasma detection by culture are difficult and may take as long as five weeks to generate results which may be inconclusive or inaccurate. Mycoplasma may also be detected by the presence of antibodies directed against mycoplasma species. although this assay has a rapid turnaround time, it may lack sensitivity and specificity. Molecular methods such as DNA probes and polymerase chain reaction (PCR) techniques have also been used to detect Mycoplasma (Rasin et al., Mol. Cell. Probes 8:497-511, 1994; van Kuppeveld et al., Appl. Environ. Microbiol. 58:2606-2615, 1992; Hopert et al., J. Immunol. Meth. 164:91-100, 1993).
The practical use of PCR has been extended to multiple primer systems to meet the increased demand for multi-species detection assays (Wang et al., Mol. Cell. Probes 11:211-216, 1997; Kulski et al., J. Clin. Microbiol. 33:668-674, 1995). Multiplex PCR allows for the simultaneous detection and differentiation of multiple species with a high level of sensitivity and specificity.
There is an ongoing need for methods of identifying the three mycoplasma species mentioned above, and for detecting CFS infection. The present invention addresses this need.
One embodiment of the present invention is a method for determining an increased likelihood of the presence of chronic fatigue syndrome (CFS), fibromyalgia (FMS) or rheumatoid arthritis (RA) in an individual, comprising the steps of: isolating peripheral blood mononuclear cells (PBMC) from the individual; and detecting the presence of at least one mycoplasma species in said PBMC, wherein the presence of at least one of these species indicates an increased likelihood of the presence of CFS, FMS or RA. In one aspect of this preferred embodiment, the species is M. fermentans, M. hominis or M. penetrans. Preferably, the detecting step comprises a polynucleotide amplificaiton reaction. More preferably, the detecting step comprises multiplex PCR. Alternatively, the detecting step comprises Southern hybridization or dot blot hybridization. In one aspect of this preferred embodiment, the amplification reaction comprises use of two or more oligonucleotide primers selected from the group consisting of the sequences shown in SEQ ID NOS: 3-8. Preferably, the primers shown in SEQ ID NOS: 3-8. In one aspect of this preferred embodiment, the amplification reaction comprises use of two or more oligonucleotide primers having sequences shown in SEQ ID NOS: 3 and 4 so as to amplify a 206 base pair region of M. fermentans DNA. In another aspect of this preferred embodiment, the amplificaiton reaction comprises use of the primers having sequences shown in SEQ ID NOS: 5 and 6 so as to amplify a 170 base pair region of M. hominis DNA. In another aspect of this preferred embodiment, the amplification reaction comprises use of the primers having sequences shown in SEQ ID NOS: 7 and 8 so as to amplify a 407 base pair region of M. penetrans DNA. Preferably, the detecting step comprises detecting two or more mycoplasma species. Advantageously, the two or more species are selected from the group consisting of M. fermentans, M. hominis and M. penetrans. Preferably, all three of these species are detected. Preferably, the two or more species are simultaneously detected. More preferably, all three species are simultaneously detected.